Make the PCR template as follows:
Mix the LtrB-IBS template (20 ng/μl) with the LtrB-EBS template (20 ng/μl).

LtrB-IBS template is prepared by PCR using the LtrB-IBS-F primer, EBS Universal primer, and pACD4 LtrB intron donor plasmid as a template. Gel purify the PCR product to remove the plasmid template. Dilute to concentration of 20 ng/μl.

LtrB-IBS-F            5'- GTG CGC CCA GAT AGG GTG TTA AGT C -3'
EBS Universal primer  5'- AAT TAG AAA CTT GCG TTC AGT AAA CAC AAC TTA TAC -3'

Final PCR product (216 bp):

1801                             gtgc gcccagatag ggtgttaagt caagtagttt 
1861 aaggtactac tctgtaagat aacacagaaa acagccaacc taaccgaaaa gcgaaagctg 
1921 atacgggaac agagcacggt tggaaagcga tgagttacct aaagacaatc gggtacgact 
1981 gagtcgcaat gttaatcaga tataaggtat aagttgtgtt tactgaacgc aagtttctaa 
2041 tt

LtrB-EBS template:
Mix the LtrB-EBS-T primer with the LtrB-EBS-B primer and dilute to concentration of 20 ng/μl.

LtrB-EBS-T  5'- CGA TAG AGG AAA GTG TCT GAA ACC TCT AGT ACA AAG AAA GGT AAG TTA -3'
LtrB-EBS-B  5'- TAA CTT ACC TTT CTT TGT ACT AGA GGT TTC AGA CAC TTT CCT CTA TCG -3'

Make the LtrB primer mix as follows:
22.0 μl of water
2.0 μl of EBS2 primer (10 μM)
2.0 μl of EBS Universal primer (10 μM)

EcI5 TargeTron

Make the PCR template as follows:
Mix the EcI5-IBS template (20 ng/μl) with the EcI5-EBS template (20 ng/μl).

EcI5-IBS template is prepared by PCR using the EcI5-IBS-F primer, EcI5-IBS-R primer, and pACD3 EcI5 intron donor plasmid as a template. Gel purify the PCR product to remove the plasmid template. Dilute to concentration of 20 ng/μl.

EcI5-IBS-F   5'- GTG CGA CAT GAA GTC GCC TGA ATA ATT G -3'
EcI5-IBS-R   5'- TAA CGA CGC TTC AGC AGT TCA CAT ATG -3'

Final PCR product (252 bp):

1801                         gtgcgaca tgaagtcgcc tgaataattg ttccagcgga 
1861 gttcgattcc gtcagggaac ctgatgttcc gtcatcagta gcctactgac acatgcgtca 
1921 ctggtaacgg tggggtgtga agctgtcagg acaatgaaac cggatcttcg gatcgcatga 
1981 aaccgtgagg ttacatgtaa tctgccagca tcaggcggag gaggtctagg ctcggtagca 
2041 tgactaacat atgtgaactg ctgaagcgtc gtta

EcI5-EBS template is prepared by PCR using the EcI5-EBS-X-F primer (where X corresponds to G, A, T, or C according to the desired EBS3 nucleotide residue), EcI5-EBS-R primer, and pACD3-EcI5X intron donor plasmid (where X corresponds to G, A, T, or C according to the desired EBS3 nucleotide residue) as a template. Gel purify the PCR product to remove the plasmid template. Dilute to concentration of 20 ng/μl.

EcI5-EBS-G-F  5'- CTA GGG GTA CAC GGA CAA TAA ACC ACC GGT GTT GTG AG -3'
EcI5-EBS-A-F  5'- CTA GGG GTA CAC GGA CAA TAA ACC ACC GGT GTT ATG AG -3'
EcI5-EBS-T-F  5'- CTA GGG GTA CAC GGA CAA TAA ACC ACC GGT GTT TTG AG -3'
EcI5-EBS-C-F  5'- CTA GGG GTA CAC GGA CAA TAA ACC ACC GGT GTT CTG AG -3'

EcI5-EBS-R  5'- TAT CCG GTC CAT TAC AGA CTG GCA TTC -3'

Final PCR product (186 bp):

2101                                               ctaggggt acacggacaa 
2161 taaaccaccg gtgttgtgag cagagtctaa cctactgttg ttattcaggt ggaacatggt 
2221 aagcccgtat cgctgcctcg tgaggcaggt taaccgcaag ggatactgtt ggcggtgcgg 
2281 gtaaaagaag gtggaaaaag cgaatgccag tctgtaatgg accggata

Make the EcI5 primer mix as follows:
22.0 μl of water
2.0 μl of EBS1S primer (10 μM)
2.0 μl of EBS2AS primer (10 μM)